Pseudomonas aeruginosa is an opportunistic pathogen which causes severe and often fatal infections in compromised or immunosuppressed individuals. Among the various extracellular products secreted by P. aeruginosa, exotoxin A (a potent inhibitor of eukaryotic protein synthesis) is the most toxic, and available evidence suggests that this toxin is a major virulence factor. Available evidence indicates that active or passive immunization against exotoxin A may be of value in the prophylaxis and treatment of infections with P. aeruginosa. Support is requested for the preparation of non-toxic forms (toxoids) of P. aeruginosa exotoxin A, to be used for vaccine production. The approach involves combining recombinant DNA techniques with a novel photochemical reaction recently discovered in this laboratory, and will include (i) photochemical identification of a residue (or residues) within the catalytic center of the toxin or of cloned toxin fragments; (ii) construction of enzymically inactive mutant toxins in Escherichia coli by site-directed or deletion mutagenesis of active site residues; (iii) development of conditions for production and purification of these inactive forms of exotoxin A; and (iv) biochemical and biological characterization to identify those non-toxic constructs which retain both immunogenicity and antigenicity. By selecting specific amino acid substitutions, based upon photochemical data and the known three-dimensional structure, potential reversion of non-toxic constructs, as well as alterations of secondary and tertiary structure, will be minimized. Ideally suited to the case of exotoxin A, this approach may prove generally useful in the construction of effective toxoids for other ADP-ribosylating exotoxins.